Oligonucleotide Repair of a Retinal Degeneration Model

描述（申请人提供）：编码视杆细胞cGMP磷酸二酯酶（β-PDE）β亚基的基因突变与视网膜色素变性（RP）相关，RP是一种导致人类失明的疾病家族。小鼠rd 1（Pde 6 b rd 1）和rd 10（Pde 6 b rd 10）视网膜变性表型是研究人RP的模型，因为这些表型是由编码β-PDE的基因突变引起的。寡核苷酸指导的基因校正是一种策略，其中治疗性寡核苷酸与其基因组靶序列之间的错配被内源性DNA修复机制识别，诱导它们将突变转化为野生型。与其他基因疗法相反，这种策略旨在永久修复染色体基因中的缺陷。这项拟议研究的主要目的是确定是否可以使用阿托伐他汀指导的基因校正来修复rd 1或rd 10突变，防止感光细胞变性和保护视力。在特异性目的1中，将通过免疫组织学和免疫印迹法确定是否存在阿托伐他汀诱导的DNA修复蛋白。将使用无细胞测定来确定寡核苷酸是否在视网膜和感光蛋白存在下诱导测试DNA靶标的修复。相同的方案将与单个修复蛋白的抑制剂结合，以测试它们在修复反应中的重要性。在特定目标2中，将寡核苷酸注射并离子电渗至新生小鼠的眼睛中。将通过免疫印迹和免疫组织化学检查视网膜中的视紫红质和β-PDE来评估光感受器拯救。由于视紫红质是表达最丰富的感光细胞标志物，监测其水平应该提供感光细胞拯救的灵敏和可靠的评估。通过对视网膜进行β-PDE免疫组织化学检查和测定视网膜提取物的PDE 6酶活性来评估DNA修复。视觉功能的非侵入性测量（例如，ERG）也将被采用。在这些动物模型中预防视力丧失应该指导我们对遗传性视网膜疾病如人类常染色体隐性遗传性RP的基因治疗的努力。

DESCRIPTION (provided by applicant): Mutations in the gene encoding the beta subunit of rod photoreceptor cGMP phosphodiesterase (beta-PDE) are associated with retinitis pigmentosa (RP), a disease family that leads to blindness in humans. The mouse rd1 (Pde6b rd1) and rd10 (Pde6b rd10) retinal degeneration phenotypes are models for the study of human RP because these phenotypes result from mutations in the gene encoding beta-PDE. Oligonucleotide-directed gene correction is a strategy in which a mismatch between a therapeutic oligonucleotide and its genomic target sequence is recognized by endogenous DNA repair mechanisms, inducing them to convert the mutation to wild type. As opposed to other gene therapies, this strategy is intended to permanently repair the defect in the chromosomal gene. The broad objective of this proposed research is to determine whether oligonucleotide-directed gene correction can be used to repair the rd1 or rd10 mutations, prevent photoreceptor degeneration, and preserve vision. In Specific Aim 1, the existence of oligonucleotide-inducible DNA repair proteins will be determined by immunohistology and immunoblotting. A cell-free assay will be used to determine whether oligonucleotides induce repair of test DNA targets in the presence of retina and photoreceptor proteins. The same protocol will be coupled with inhibitors of individual repair proteins to test for their importance in the repair reaction. In Specific Aim 2, oligonucleotides will be injected and iontophoresed into eyes of neonatal mice. Photoreceptor rescue will be assessed by immunoblotting and immunohistochemical examination of retinas for rhodopsin and beta-PDE. Since rhodopsin is the most abundantly expressed photoreceptor marker, monitoring its levels should provide an exquisitely sensitive and robust assessment of photoreceptor rescue. DNA repair will be assessed by immunohistochemical examination of retinas for (-PDE and by assaying retinal extracts for PDE6 enzymatic activity. Noninvasive measures of visual function (e.g., ERG) will be taken also. Preventing vision loss in these animal models should direct our efforts for gene therapy of inherited retinal diseases such as autosomal recessive RP in humans.